ABSTRACT Our goal is to develop and apply chemical biological methodologies that will assist in the identification and characterization of gut microbiome enzymes responsible for the degradation of the gut mucus. The intestinal tract harbors an enormous and diverse collection of commensal bacteria, termed the gut microbiome, that are essential for metabolism, immune development and homeostasis, and epithelial cell angiogenesis. In healthy individuals, the majority of microbes are held at bay in the gut lumen by the steady secretion of mucus, which primarily consists of crosslinked and heavily O-glycosylated mucin proteins. In inflammatory bowel diseases (IBD), including ulcerative colitis and Crohn?s disease, the gut microbes manage to infiltrate the host intestinal mucin protein core, epithelium, and lamina propria. The resulting recognition of microbial constituents by the host?s innate and adaptive immune responses drives the intestinal inflammation that is associated with all forms of IBD. We posit that progression from a healthy state to colitis commences via microbial erosion of the mucus layer and that colitis-associated gut microbes, including pathogenic and infectious bacteria and viruses (i.e., C. difficile, cytomegalovirus), harbor aberrant protein functionalities that are far more adept at host mucus layer degradation than microbes found in healthy individuals. Our hypothesis is supported by recent discoveries that, in murine models of colitis, elevated levels of sialic acid-degrading enzymatic activity are found. In this proposal, we will focus on elucidating microbial proteins that recognize and degrade sialic acid, an abundant and the most terminally expressed carbohydrate on mucus glycans. We believe that, while the many layers of mucus glycans beneath sialic acid aid in microbial defense in healthy individuals, removal of sialic acid is the first overall step in microbial infiltration of the gut. To test this hypothesis, we have developed UV-photoactivatable sialic acid-based chemical probes that irreversibly label all host and bacterial proteins from microbiome samples capable of binding sialic acid (i.e., sialidases, lectins, transporters, transferases). All probe-bound proteins are subjected to affinity-based enrichment and subsequent protein identification and quantitation with mass spectrometry (MS). Here, we will employ our sialic acid probes and innovative methods to a well-established adoptive T cell transfer murine model of chronic colitis and directly compare our metaproteomics results to those obtained from control mice raised under identical conditions. Differences observed between healthy and colitic mice may implicate which enzymes are critical for human IBD and assist in achieving our long-term goal of identifying microbial proteins that promote colitis. Importantly, we are well qualified to accomplish our proposed aim, as the Wolan laboratory spearheaded the development and application of chemical probes and MS-based metaproteomics to find the bacterial cysteine proteases that are overly abundant in colitic microbiomes. The probes, methods, and results generated here will lead to exciting new opportunities for studying host-microbe interactions.